The present invention relates to a method and system for processing scan-data from a confocal microscope. More specifically, the invention relates to a method and system enabling real-time processing of the scanned data.
In confocal microscopy a specimen is scanned with a focused laser beam. The focus of the laser beam is moved in a section plane of a specimen by two scan mirrors tilting around their respective axes, which axes are perpendicular to each other. The first scan mirror diverts the laser light in the x-direction and the second scan mirror diverts the laser light in the y-direction while the intensity of the reflected or the fluorescent light is measured for each scanning point. Each measured intensity value relates to an x, y and z-position of the specimen, therefore, providing a user with a three-dimensional image of the specimen.
An example for a confocal microscope is disclosed in U.S. Pat. No. 5,804,813 xe2x80x9cDifferential confocal microscopyxe2x80x9d to J. P. Wang et al. That Patent describes a Hexe2x80x94Ne laser as the light source, and a microscope objective lens as a focusing device. The light signal reflected from the surface of a specimen travels through a beam splitter and is almost completely reflected at the surface to travel to an optical detector which can use photodiodes, avalanche photodiodes, photo multipliers, charge coupled devices (CCDs), or fluorescent screens. The signal is detected by the optical detector and then amplified by a signal amplifier. The amplified signal is recorded by an analog-to-digital converter and then stored in a computer. The computer generates a three-dimensional image by using the intensity of the signal corresponding to the respective coordinates of the specimen. Before the measurement is performed, it is necessary to use the same sample to calibrate the relationship between the variation of signal intensity and the height of the sample.
The ideal scan pattern of the light beam on the surface of a specimen is a meander pattern. Such a scan pattern is usually generated by scanning one line in the x-direction (a xe2x80x9c+xe2x80x9dx-direction) with a constant y-position, then stopping the scan in the x-direction and moving the y-position of the beam to the next scan line, then scanning this new line in the opposite x-direction (a xe2x80x9cxe2x88x92xe2x80x9d x-direction) and so on. In reality, however, the meander scan pattern does not exist for high scan rates due to the inertia of the galvanometric devices and scanning mirrors. The real path of the scanning beam at scan rates higher than 100 Hz is a sinusoidal curve which requires a correction to the ideal path. Several types of errors may occur during scanning due to, for example, a higher velocity of scanning at a turning point of the real path or due to a different shape of the scan path for different scan directions. In addition, different run and process times for the intensity and position signals have to be considered.
Registration and processing of a position signal is normally done with analog circuit, computers or digital signal processors (DSP). There are certain disadvantages of signal processing with analog circuits. For example, signal processing can only be carried out with the help of a correction function implemented in an analog circuit. Changes of the correction mechanism, such as for example, changes of a scan rate, require a lot of effort. Moreover, the accuracy of analog circuits with respect to mathematical operations reaches its limit at high scan rates. Pixel rates greater than 1 MHz at a 12-bit accuracy can be achieved only with enormous effort.
Signal processing with a computer or DSP is possible at low scan rates. However, while signal processing is flexible because of the ease with which the algorithms used in such processing can be changed or modified, at higher scan rates computers or DSPs fail due to their inability to perform data processing in real time.
It is, therefore, an object of the present invention to provide a system for real time processing of digital scan-signals from a confocal microscope, in which system the accuracy of the measurements depends solely on the accuracy of detectors and analog-to-digital converters. Furthermore, the processing of the scan-signals becomes possible at high scanning rates. This object of the invention is accomplished by a system having at least three different analog signals generated by a confocal microscope, at least three analog-to-digital converters each receiving a different analog signal and producing a digital signal at an early stage of processing, and a control and processing unit which uses a plurality of programmable devices to process the digital signals in real time.
It is also an object of the present invention to provide a confocal microscope with a system for real time processing of scan signals at high scanning rates. Additionally, an object is to provide a flexible system allowing a user to easily modify existing algorithms.
This object is accomplished by a confocal microscope comprising an illumination source, a scanning device with a scanning mirror system, a control and processing unit, which uses a plurality of programmable devices for processing the digital signals in real time, the control and processing unit having at least three input ports and one output port, a first detector generating analog signals corresponding to the light reflected from a specimen, a second detector generating analog signals corresponding to the intensity of the light from the illumination source, an electrical connection providing the control and processing unit with an analog position signal generated by the scanning device, and a first, a second and a third analog-to-digital converters for digitizing the analog signals received from the first and second detectors and the scanning device.
Yet another object of the present invention is to provide a method for real time processing of scan-signals from a confocal microscope, the method enabling the processing of the analog scan-signals at high scanning rates and loss free data collecting.
This object is achieved by a method comprising:
generating at least three different analog signals by a confocal microscope, wherein a first signal corresponds to the light reflected from a specimen, a second signal corresponds to an illumination reference and a third signal (a position signal) corresponds to a position of light on the specimen;
converting each analog signal into a respective digital signal by using a separate analog-to-digital converter for each analog signal;
directing the digital signals to a control and processing unit which uses at least one of programmable devices for processing the digital signals in real time;
buffering the digital position signals in a line buffer of the control and processing unit;
correcting the first digital signal for intensity fluctuations of the second digital signal to generate a corrected digital detection signal; and
combining the corrected digital detection signal with a digital position signal to provide a combined signal.
An advantage of the inventive method and system is that the position signal of the focused scanning beam and of the signals from the detectors (the scanning beam and reference beam) are digitized at a very early stage. Processing of the data is done mainly in a digital form with the use of a programmable control and processing unit which is implemented in a programmable digital circuit, such as, for example, FPGA (Field Programmable Gate Array). Correction parameters can be used online, making the subsequent image processing much easier or unnecessary at all. The accuracy of the device depends solely on the accuracy of the detectors and of the analog-to-digital converters. Analog-to-digital converters with a large processing bandwidth are available at reasonable costs. The position correction function depends on the measurement condition variables and can be easily changed or modified online. The dynamic pixel accumulation allows the system to record several measurement values during the scan of an image pixel and to accumulate the data in a suitable buffer. The dynamic pixel accumulation during a measurement allows the system to operate in a wide dynamic range of scanning rates from a few Hz to up to 250 MHz.
Pixel accumulation calls for a simple analog circuit without giving up the high resolution and wide dynamic range. Despite the fact that analog-to-digital converters sample the intensity of signals only from a narrow band of signal heights, repeated scanning and pixel accumulation result in a theoretically unlimited bit resolution. The pixel accumulation technique also allows one to adjust the time necessary for data processing to the speed of a scan. For example, during a slow scan measurement the detected intensity signals are accumulated until the scanning beam reaches the next pixel element of the object. The pixel accumulation rate can then be adjusted to the data processing rate.
The advantages of the pixel accumulation method implemented in the present invention are similar to those of the line accumulation method, in which the intensity data obtained from multiple sequential scans of one scan line are stored in a suitable buffer arrangement. Differences between the line scans in different directions are averaged out by repeated line accumulation. In case of resonant galvanometers for the x-direction scans, only the line accumulation method is used, since pixel accumulation is not possible.
A further advantage of the system is its ability to compensate intensity fluctuations in real time, which repeats the predetermined process steps with the accuracy up to nanoseconds. The system of the present invention satisfies the requirements of flexibility and real time data processing, because, for example, there is no need to buffer data, since a computer controlled data recording is not used. The real time processing ability of the inventive system provides a loss free and flexible data collection with the highest possible scan rates. Computers are controlled by interrupts, so a running process has to be ended before further data processing can go on. The present invention has the ability of a high flexibility with respect to data processing changes and real time processing of the data.